Given the variety of physiological parameters, including ion conductances, that are affected by changes in intracellular pH (pH), the regulation of pH is expected to be especially important for neurons. I propose to continue studies on two important aspects of acid-base transport in two model excitable cell systems, the squid giant axon and the giant barnacle muscle fiber. In the squid axon, we will characterize the process by which pH recovers from intracellular alkaline loads; our preliminary work suggests that this pH recovery is mediated by a novel K/HCO3 cotransporter. In barnacle muscle, we will examine the signal transduction mechanism by which cell shrinkage leads to an activation of the Na-H exchanger. Some of these Na-H exchanger experiments are better suited to Xenopus oocytes, which we have recently shown to possess a shrinkage-activated Na-H exchanger. For both the squid and barnacle preparations, we will use the internal dialysis technique to control the intracellular milieu, and use pH-sensitive microelectrodes to monitor pH. We will computer fluxes mediated by the transporters from rates of pH change (dpH/dt) change and the measured intracellular buffering power. For the squid-axon work (don in Woods-Hole during annual 6-week experimental periods), we will characterize the putative K/HCO3 cotransporter using pH measurements, as well as measurements of 86Rb+ and/or 42K+ fluxes. We will quantitate the putative transporter's dependence on pH and pH, its dependence on [K+], the ability of other cations to replace K+, the dependence on [HCO3], the requirement for ATP, and the cation fluxes that accompany the pH changes. This putative K/HCO3 contransporter could be the elusive mechanism responsible for "background acid loading" in a wide variety of cells. The power of the internally dialyzed squid axon is uniquely amenable to the initial characterization of this transporter. The work on barnacle muscle fibers and oocytes we will characterize the shrinkage-induced activation of Na-H exchange using pH measurements, as well as microinjections of substances such as cholera toxin, purified constitutively-active G-protein alpha subunits, and antisense nucleotide sequences designed to knock out specific alpha subunits. We will (i) determine if shrinkage increases the rate at which GTPgammaS permanently activates the Na-H exchanger; (ii) determine which G-protein alpha subunit(s) lead to shrinkage-induced activation of the Na-H exchanger; (iii) explore in BMFs the possibility that shrinkage activates Na-H exchange via a phosphorylation; (iv) explore the interaction between the shrinkage and pH pathways for activating Na-H exchange. This proposed work will provide fundamental information concerning specific acid-base transporters and how they are controlled, and has broad implications for cellular physiology.